The nitrogen-fixing bacterium Gluconacetobacter diazotrophicus, previously known as Acetobacter diazotrophicus (Gillis, M. et al. Int. J. Syst. Bacteriol. 39:361-364; 1989), was originally isolated from within sugarcane roots and stems (Cavalcante, V. A., et al. (1988) Plant Soil Vol. 108, p. 23-31). It has been demonstrated by 15N2 incorporation that G. diazotrophicus fixes nitrogen inside sugarcane plants (Sevilla, M. et al. Mol. Plant Microbe Interact. 14:358-366; 2001; Boddey, R. M. et al. Plant Soil 252:139-149; 2003) and that it has a capability to excrete almost half of the fixed nitrogen in a form that is potentially available to plants (Cojho, E. H et al. Fed. Eur. Microbiol. Soc. Microbiol. Lett. 106:341-346; 1993). The bacterium invades between cells of sugarcane root meristems and at emergence points of lateral roots colonizing intercellularly, and also in the xylem, without nodulation (James, E. K. et al. J. Exp. Bot. 52:747-760; 2001). The conditions under which intracellular colonisation of Gd could occur enabling non-nodular endosymbiotic nitrogen fixation has been demonstrated (EP-B-1422997 and Cocking, E. C., et al. (2006) In Vitro Cellular and Developmental Biology—Plant Vol. 42, No. 1, p 74-82). In particular, the bacteria are administered to the growth medium of the plant as the plant grows on germination or within 7 days thereof.
WO2011/144741 suggests that bacteria such as Gd, may be injected into stems of sugarcane to enhance nitrogen-fixation. Clearly such a technique is not one which could be applied in any large scale agricultural operation.
The applicants have found that growing plants can be successfully inoculated with nitrogen fixing bacteria.